Among the problems encountered in conventional drug use today are loss of hair, diarrhoea and suppression of the immune response system in cancer chemotherapy, persistence of intracellular microbial infections in anti-microbial therapy, accumulation of toxic metals in tissues, and ineffectiveness of many vaccines. Indiscriminate drug action against target and normal cells, inability of drugs to penetrate body areas requiring treatment, and premature drug elimination or inactivation can explain some of these effects.
Approximately twenty five years ago it was discovered that mixing dry phospholipids with water results in the spontaneous formation of spherules containing bilayers of phospholipid molecules resembling biological membranes. As these spherules form, they entrap any water soluble substances that may be present into vesicular voids. Lipid-soluble substances, on the other hand, are incorporated into the liposomal membrane. Because incorporation into the aqueous or lipid phase is passive, no special methods have to be tailored for various substances to be incorporated. Virtually any substance, regardless of charge, solubility or other characteristics, can be incorporated into liposomes.
Liposome instability can arise when stored vesicles collide and fuse to form large liposomes. In vivo, these large vesicles will be removed from the circulation much more rapidly than small liposomes. This fusion of small liposomes can be reduced by imparting a net negative charge to the spherules. Drug loss due to leakage from liposomes can be reduced by incorporating excess cholesterol into the membrane.
Liposomes have been contemplated as being be a promising means for encapsulating a variety of substances to be delivered in vivo to patients, including drugs useful in cancer chemotherapy. For liposomes to be beneficially employed in these situations, it is necessary that they be manufactured in specific shapes and sizes, and also maintain stability during subsequent administration. Thus, there has been, and continues to be, considerable effort expended toward the development of suitable methods for producing liposomes having these medically useful properties.
There exist at least three classes of liposomes; unilamellar liposomes, multilamellar liposomes, and multivesicular liposomes. Multivesicular liposomes are spheroids having multiple, separate internal compartments formed from septa of lipid bilayers. Unilamellar liposomes consist of single spheroid bilayers. Multilamellar liposomes, on the other hand, are spheroids having multiple concentric bilayers. Bangham, et al. in the Journal of Molecular Biology, Volume 13, pages 238-252 (1965), described some of the general methods for making multilamellar liposomes as well as their properties. Huang in Biochemistry, Volume 8, pages 344-352 (1969), described unilamellar liposomes. Similarly, Kim et al. in Biochem. Biophys. Acta., Volume 646, pages 1-10 (1981), described a novel method for producing unilamellar liposomes. The properties and problems associated with manufacturing multivesicular liposomes were also described by Kim et al. in Biochem. Biophy. Acta., Volume 782, pages 339-348 (1983).
In addition to the above references, numerous other references describing methods for producing non-multilamellar liposomes exist. These include U.S. Pat. Nos. 4,078,052 and 4,235,871, issued to Papahadjopoulos; U.S. Pat. No. 4,224,179, issued to Schneider; U.S. Pat. No. 4,310,506, to inventor Baldeschwielder; and U.S. Pat. No. 4,522,803, issued to Lenk.
In U.S. Pat. No. 4,394,372, inventor Taylor discloses a method for making unilamellar liposomes. This method also yields some form of contaminating multilamellar structures not well described by the patentee. A shortcoming of the procedure for production of multilamellar structures is that the size distribution of multilamellar structures in Taylor's method is not adjustable, thus decreasing the flexibility of using this type of liposome as a drug delivery vehicle.
In addition to the above drawbacks, the Taylor method has the limitation that it is not specifically designed for making multilamellar liposomes. Furthermore, the process requires the use of a narrowly defined two-compound organic solvent system where one solvent is hydrophilic and the other is hydrophobic. The former must have a high partition coefficient in aqueous solutions, but must not dissolve in the latter. The latter must not totally dissolve the lipid membrane components by itself, but the membrane components must be completely soluble in the mixed solvent system. Together, the component system must form an interface with the aqueous solution.
Methods of preparing multilamellar liposomes are described in the literature. However, all of these methods have limitations which restrict their successful implementation in the medical arena. Perhaps the best known method is that of Bangham et al., Journal of Molecular Biology, Volume 13, pages 238-252 (1965). This method consists of first solubilizing a suitable lipid composition in an organic solution, then forming a dry layer of the lipid composition by removing the organic solvent, generally under vacuum, and hydrating the lipid composition using a suitable aqueous solvent followed by mechanically forming multilamellar liposomes with a sonicator or vortexer to disperse the semi-solid lipid particles. A modification of this method, described in U.S. Pat. No. 4,485,054, consists of depositing the lipids on the surface of glass beads or other inert materials. Subsequent agitation more efficiently forms multilamellar liposomes.
In addition to the above, U.S. Pat. No. 4,308,166, issued to Marchettei, describes the preparation of liposomes by the addition of a phospholipid emulsion to a lyophilized, or otherwise dried, active substance. In U.S. Pat. No. 4,508,703, inventor Redziniak describes a method of preparation of multilamellar vesicles comprising atomizing lipids by dissolving them in a volatile organic solvent. The lipids are sprayed onto a suitable surface and then hydrated to form liposomes. Finally, Szoka, et al., Annual Review of Biophysics and Bioengineering, Volume 9, pages 467-508 (1980), presents a comprehensive review of methods and problems associated with the production of liposomes.
Although a survey of the literature makes it apparent that there are numerous ways of preparing liposomes having different physical properties, it is also apparent that each of these methods, and the liposomes produced, have certain technical drawbacks that prevent implementing them in the medical arena as vehicles for transporting drugs to particular body sites. Perhaps the major difficulty, particularly related to the generation of multilamellar liposomes, is the inability to precisely and reproducibly control the average multilamellar liposome size over a wide range. As alluded to above, the size of the liposome is crucial in many instances.
Although numerous attempts have been made to solve the above mentioned shortcomings, the efficiency and predictability required for medical applications has thus far not been achieved.